Patent DE-102005045560-A1 (DE)
Title: Quantitative determination of absolute allele copy number per cell, comprises accomplishing PCR, determining count of different PCR products and comparing the count of different PCR products using probability distribution
Inventor: GAUER CHRISTOPH
IPC: C12Q1/68
Filed: 2005-09-23
Abstract: The quantitative determination of absolute allele copy number per cell by determining count of pre-determined nucleic acid sequence and a biological sample homologous to pre-determined sequence, comprises accomplishing PCR, determining count of different PCR products and comparing the count of PCR products using probability distribution. The PCR-reaction(s) is/are adapted to amplify two non-homologous sequences, which are comprised in the pre-determined sequence and which are short tandem repeats-, variable number of tandem repeats- and single nucleotide polypeptide segments. The quantitative determination of absolute allele copy number per cell by determining count of pre-determined nucleic acid sequence and a biological sample homologous to pre-determined sequence, comprises accomplishing PCR, determining count of different PCR products and comparing the count of PCR products using probability distribution. The PCR-reaction(s) is/are adapted to amplify two non-homologous sequences, which are comprised in the pre-determined sequence and which are short tandem repeats-, variable number of tandem repeats- and single nucleotide polypeptide segments. The pre-determined nucleic acid sequence is a chromosome, a gene or gene segment. A quantity of the biological sample leading to an allelic dropout is provided during the execution of a PCR. The biological sample provides less than 10 pg DNA and less than 1 cell. The probability distribution is composed of the data of the average values of the different PCR-products obtained during the multiple determination procedure with the individual reference samples with a defined and different copy number of the pre-determined sequence respectively. The determination of the probability distribution requires 4-10 reference samples with defined and different copy number of the pre-determined sequence. The PCR reaction(s) is/are accomplished 100 times for each reference sample. The PCR-reaction(s) is/are adapted to amplify two non- homologous, highly polymeric sequences from the non-coding region of the DNA and the sequences occur in the genome of the donor only once per each allele. The PCR-reaction(s) is/are adapted between 5-12 of non-homologous sequences. The number of copies of the pre-determined sequence in the biological sample is 0-5. The execution of the PCR-reaction and the quantity of assigned biological sample are adapted to determine the biological sample that contains the pre-determined sequence in a copy number per cell of 0, 1, 2 or 3. The biological sample is amplified with a nonspecific PCR before execution of the reaction product that is divided into the number of partial quantities. A pair of primers is adapted to amplify two non-homologous sequences that are used in every PCR. The determination of the presence and/or absence of PCR products takes place by gel electrophoresis, hybridizing techniques on a DNA array, bead system or an optical, electrical or electro-chemical measurement. The sequence of the PCR products is determined by DNA sequencing or a hybridizing procedure. The length of the PCR products is determined by capillary electrophoresis. The parameters in the PCR are selected in such a manner that the relative probability for a positive amplification reaction for each of the two non-homologous sequences is 0.5. A pole body is used as biological sample after first meiotic division. Independent claims are included for: (1) kit for the quantitative determination of absolute allele copy number per cell; and (2) a device for the quantitative determination of absolute allele copy number per cell.